The broad objective of this work is to contribute to the understanding of how DNA replication is regulated in Escherichia coli. More specifically the aim of this research is to determine the role of the dnaA gene product and other DNA binding proteins in the initiation of DNA replication. Evidence exists from other laboratories that the dnaA product is a 50-54 Kd protein which may be involved in the regulation of initiation by some interaction with RNA polymerase which may allow either an RNA primer to be made at the origin of replication or allow transcriptional activation of initiation. The dnaA protein may also regulate its own synthesis. This laboratory has isolated a phenotypic revertant of a dnaA mutant, E508-R1. This phenotypic revertant can initiate replication at 42 degrees and overinitiates replication at 32 degrees. This strain may be defective in regulating its own synthesis as it appears to over-produce a 50-53 Kd protein, which may be the dnaA product. Other DNA binding proteins, including the beta and beta'-subunits of RNA polymerase, also appear elevated in this revertant. These proteins can be isolated by DNA-cellulose and heparin-agarose chromatography, and by preparative polyacrylamide gel electrophoresis. The identification will be made by SDS polyacrylamide gel electrophoresis. Using these methods it should be possible to isolate large enough quantities of the above proteins for characterization. It is the intent to determine whether the 50-53 Kd protein which is over-produced in the phenotypic dnaA revertant is indeed the dnaA product (as described by other laboratories) by comparison of the proteins by O'Farrell gels, partial proteolysis and antibody binding. In order to gain more information on the possible regulatory role of the dnaA product and other DNA-binding proteins, the interaction of these proteins with RNA polymerase will be examined by antibody precipitation experiments. Also the possible interaction of these proteins with the origin site on DNA will be investigated by testing for specific binding to a small plasmid which carries the origin of replication.